The applicant, a D.V.M. with some clinical and laboratory experience, seeks support to become an independent researcher in the field of viral pathogenesis and vaccine development. His long-term goal is to better understand the pathogenesis of human alphaviral encephalitides, including the roles of each viral gene. This information will be used to develop safer and more effective vaccines and therapeutics. The proposed research focuses on Venezuelan equine encephalitis virus (VEEV), an important, naturally emerging human pathogen and a potent biological weapon categorized by the NIAID as a Category B Priority Pathogen. Although VEEV is an important human pathogen and an efficient vaccine expression vector, little is known about the role of the nonstructural proteins (replicative machinery) of this virus in pathogenesis. Furthermore, no licensed human VEEV vaccine is available. The goals are to1l) develop safe and effective VEEV vaccines using chimeric VEEV/Sindbis viruses (SINV) that are efficiently produced at large scale, and; 2) study VEEV pathogenesis using reciprocal SINV/VEEVchimeras to assess the roles of structural vs. nonstructural proteins in tissue tropism and pathogenicity. Two central hypotheses will be addressed: 1) The virulence potential of VEEV strongly depends on the nonstructural proteins and probably their interactions with cellular/organismal factors, and; 2) VEEV tissue tropism depends on the ability of the virus not only to bind and enter target cells, but also to establish productive infection. Better understanding of these VEEV-cell interactions will allow the applicant to generate highly attenuated SINV/VEEV chimeras that induce protective immunity against VEEV. The specific aims are: 1. Characterize the replication in vitro and in vivo, pathogenicity and immunogenicity of chimeric SINV/VEEV vaccine candidates in mice. 2. Determine the tissue tropism and pathogenicity of reciprocal, chimeric SINV/VEEV viruses in mice. Through this project, the applicant will create SINV/VEEV chimeric vaccine candidate viruses that are highly attenuated, immunogenie and replicate efficiently in cell culture. By dissecting the nonstructural proteins of VEEV from the structural proteins, the role of replieative machinery (nonstructural proteins) in the pathogenesis will be elucidated.